Journal: Nature Communications

Article Title: Inhibition of mitochondrial complex I reverses NOTCH1 -driven metabolic reprogramming in T-cell acute lymphoblastic leukemia

doi: 10.1038/s41467-022-30396-3

Figure Lengend Snippet: Panel of antibodies used for CyTOF staining.

Article Snippet: 156 Gd , p-p38 (180/182) , D3F9 , DVS-Fluidigm , 3156002A , YES.

Techniques: Staining, Marker

Journal: Cells

Article Title: Intracellular Angiotensin II Stimulation of Sodium Transporter Expression in Proximal Tubule Cells via AT 1 (AT 1a ) Receptor-Mediated, MAP Kinases ERK1/2- and NF-кB-Dependent Signaling Pathways

doi: 10.3390/cells12111492

Figure Lengend Snippet: The roles of the MAP kinase and NF-κB signaling pathways in mediating ECFP/Ang II-induced NHE3 expression in wild-type and Agtr1a -/- mPCT cells. Panel ( A ) shows that in wild-type mPCT cells, ECFP/Ang II stimulated NHE3 expression significantly, and the response was attenuated by the MEK1/MEK2 kinase inhibitor U0126 and the NF-κB activation inhibitor Ro 106–9920, respectively. However, the MEK inhibitor PD 980659 and the p38 MAP kinase inhibitor SB202196 failed to attenuate the effect of ECFP/Ang II on NHE3 expression. Panel ( B ) shows that in Agtr1a -/- mPCT cells, ECFP/Ang II failed to stimulate NHE3 expression, and the inhibitors of the MAP kinases and NF-κB signaling pathways had no significant effects on NHE3 expression. ** p < 0.01 vs. control WT mPCT cells. ++ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II.

Article Snippet: To determine the potential signaling mechanisms involved in Ad- Sglt2-ECFP/Ang II -induced biological responses, WT and Agtr1a -/- mPCT cells expressing Ad- Sglt2-ECFP/Ang II were concurrently treated with the AT 1 receptor antagonist losartan (10 μM; Tocris, Minneapolis, MN, USA), the AT 2 receptor antagonist PD 123319 (10 μM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 μM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 μM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 μM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 μM; MCE, Belleville, NJ, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Control, Transfection

Journal: Cells

Article Title: Intracellular Angiotensin II Stimulation of Sodium Transporter Expression in Proximal Tubule Cells via AT 1 (AT 1a ) Receptor-Mediated, MAP Kinases ERK1/2- and NF-кB-Dependent Signaling Pathways

doi: 10.3390/cells12111492

Figure Lengend Snippet: The roles of AT 1 and AT 2 receptors, the MAP kinases, and NF-κB signaling pathways in mediating ECFP/Ang II-induced Na + /HCO 3 - cotransporter expression in wild-type mPCT cells. Panel ( A ) shows that ECFP/Ang II significantly increased Na + /HCO 3 - expression, and the response was attenuated by losartan but not by PD123319, suggesting a dominant role of AT 1 receptors in mPCT cells. Panel ( B ) shows that the MEK1/MEK2 kinase inhibitor U0126, the NF-κB activation inhibitor Ro 106–9920, and the MEK inhibitor PD 980659 attenuated the effects of ECFP/Ang II on expression, but the p38 MAP kinase inhibitor SB202196 had no effect on Na + /HCO 3 - expression. ** p < 0.01 vs. control WT mPCT cells. ++ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II.

Article Snippet: To determine the potential signaling mechanisms involved in Ad- Sglt2-ECFP/Ang II -induced biological responses, WT and Agtr1a -/- mPCT cells expressing Ad- Sglt2-ECFP/Ang II were concurrently treated with the AT 1 receptor antagonist losartan (10 μM; Tocris, Minneapolis, MN, USA), the AT 2 receptor antagonist PD 123319 (10 μM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 μM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 μM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 μM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 μM; MCE, Belleville, NJ, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Control, Transfection

Journal: Cells

Article Title: Intracellular Angiotensin II Stimulation of Sodium Transporter Expression in Proximal Tubule Cells via AT 1 (AT 1a ) Receptor-Mediated, MAP Kinases ERK1/2- and NF-кB-Dependent Signaling Pathways

doi: 10.3390/cells12111492

Figure Lengend Snippet: The roles of AT 1 and AT 2 receptors, the MAP kinases, and NF-κB signaling pathways in mediating ECFP/Ang II-induced NF-κB, p65 expression in wild-type and Agtr1a -/- mPCT cells. Panel ( A ) shows that ECFP/Ang II increased NF-κB, p65 expression in wild-type mPCT cells, and the response was attenuated by both losartan and PD123319, supporting an important role of AT 1 and AT 2 receptors in mediating ECFP/Ang II-induced NF-κB, p65 expression in wild-type mPCT cells. Panel ( B ) shows that ECFP/Ang II alone had no significant effect on NF-κB, p65 expression in Agtr1a -/- mPCT cells, but both losartan and PD123319 potentiated this response. Panel ( C ) shows that in wild-type mPCT cells, the effect of ECFP/Ang II on NF-κB, p65 expression was attenuated by the MEK1/MEK2 kinase inhibitor U0126, the NF-κB activation inhibitor Ro 106–9920, and the MEK inhibitor PD 980659, respectively. However, the p38 MAP kinase inhibitor SB202196 had no effect on ECFP/Ang II-induced NF-κB, p65 expression in wild-type mPCT cells. ** p < 0.01 vs. control WT or Agtr1a -/- mPCT cells. ++ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II, or Agtr1a -/- mPCT cells transfected with ECFP/ANG II.

Article Snippet: To determine the potential signaling mechanisms involved in Ad- Sglt2-ECFP/Ang II -induced biological responses, WT and Agtr1a -/- mPCT cells expressing Ad- Sglt2-ECFP/Ang II were concurrently treated with the AT 1 receptor antagonist losartan (10 μM; Tocris, Minneapolis, MN, USA), the AT 2 receptor antagonist PD 123319 (10 μM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 μM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 μM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 μM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 μM; MCE, Belleville, NJ, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Control, Transfection

Journal: Frontiers in Immunology

Article Title: Gastrointestinal Microbiome Dysbiosis in Infant Mice Alters Peripheral CD8 + T Cell Receptor Signaling

doi: 10.3389/fimmu.2017.00265

Figure Lengend Snippet: Distal T cell receptor-mediated signaling is compromised in maternal antibiotic treatment (MAT) naïve CD8 + T cells . Total T cells enriched from the spleens of day of life 15 control (CTRL) and MAT infant mice were analyzed for (A) expression of Erk2 (CTRL, n = 5; MAT, n = 4), (B) percentage of phospho-Erk-1/2 (pErk1/2) positive cells (CTRL, n = 5; MAT, n = 4), (C) expression of pErk1/2 (CTRL, n = 5; MAT, n = 4), and (D) expression of c-Rel (CTRL, n = 6; MAT, n = 3) gating on naïve (CD44 − ) CD8 + T cells by flow cytometry. To analyze pErk1/2 expression (B,C) , T cells were left unstimulated (−) or stimulated (+) with anti-CD3/anti-CD28 or phorbol 12-myristate 13-acetate (PMA)/ionomycin for 2 min at 37°C. Representative graphs (CTRL, white; MAT, black; dashed line, negative staining control) and analysis of the median fluorescence intensity (MFI) or percentage are shown. Data are representative of two independent experiments, presented as mean + SEM and were analyzed by one-way ANOVA with Holm–Sidak posttest (B,C) or unpaired two-tailed Student’s t -test (D) . * p < 0.05, ** p < 0.01.

Article Snippet: Single cell suspensions of lymphocytes isolated from the spleen, MLN, or PEC of uninfected and infected infant mice were stained with optimal concentrations of the following antibodies and reagents: CD3ε (clone 145-2C11), CD8α (clone 53-6.7), CD25 (clone PC61), CD44 (clone IM7), CD62L (clone MEL-14), CD69 (Clone H1.2F3), TNF-α (clone MP6-XT22), IFN-γ (clone XMG1.2), pTyr (clone pY20), ZAP-70 (clone 1E7.2), phosphorylated ZAP-70 (pZAP-70) Tyr319 (clone n3kobu5; eBioscience), Erk2 (clone REA186; Miltenyi Biotec), phospho-Erk-1/2 (pErk1/2) Thr202/Tyr 204 (clone 4B11B69), c-Rel (clone REA397; Miltenyi Biotec), Ki-67 (clone B56; BD Biosciences), annexin V, and 7-aminoactinomycin D (7-AAD).

Techniques: Control, Expressing, Flow Cytometry, Negative Staining, Fluorescence, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Gastrointestinal Microbiome Dysbiosis in Infant Mice Alters Peripheral CD8 + T Cell Receptor Signaling

doi: 10.3389/fimmu.2017.00265

Figure Lengend Snippet: Maternal antibiotic treatment (MAT) effector CD8 + T cells do not sustain phosphorylation of Erk-1/2 . Total T cells enriched from the spleens of day of life 15 control (CTRL) and MAT infant mice were stimulated with anti-CD3/anti-CD28 for 24, 48, and 72 h and analyzed for (A) percentage of Erk2 + cells (CTRL, n = 7; MAT, n = 6), (B) expression of Erk2 (CTRL, n = 7; MAT, n = 6), (C) percentage of phospho-Erk-1/2 (pErk1/2) positive cells (CTRL, n = 7; MAT, n = 6), and (D) expression of pErk1/2 gating on effector (CD44 + ) CD8 + T cells (Teff). (E) Percentage of CD8 + Teff cells expressing low or high levels of Erk2 (Erk2 lo and Erk2 hi , respectively) and gating analysis of their corresponding pErk1/2-expressing populations at 48 h post-stimulation. Data are representative of two independent experiments and presented as mean + SEM. * p < 0.05, **** p < 0.0001, one-way ANOVA with Holm–Sidak posttest.

Article Snippet: Single cell suspensions of lymphocytes isolated from the spleen, MLN, or PEC of uninfected and infected infant mice were stained with optimal concentrations of the following antibodies and reagents: CD3ε (clone 145-2C11), CD8α (clone 53-6.7), CD25 (clone PC61), CD44 (clone IM7), CD62L (clone MEL-14), CD69 (Clone H1.2F3), TNF-α (clone MP6-XT22), IFN-γ (clone XMG1.2), pTyr (clone pY20), ZAP-70 (clone 1E7.2), phosphorylated ZAP-70 (pZAP-70) Tyr319 (clone n3kobu5; eBioscience), Erk2 (clone REA186; Miltenyi Biotec), phospho-Erk-1/2 (pErk1/2) Thr202/Tyr 204 (clone 4B11B69), c-Rel (clone REA397; Miltenyi Biotec), Ki-67 (clone B56; BD Biosciences), annexin V, and 7-aminoactinomycin D (7-AAD).

Techniques: Phospho-proteomics, Control, Expressing

Journal: Molecular and cellular endocrinology

Article Title: Estrogen receptor beta maintains expression of KLF15 to prevent cardiac myocyte hypertrophy in female rodents

doi: 10.1016/j.mce.2017.11.004

Figure Lengend Snippet: (a) TGFβ stimulates p38α activity, inhibited by SB2036580 or β-LGND. Bar graph is the mean±SD from 3 exps combined. *p < 0.05 vs. control, +p < 0.05 for TGFβ vs same + SB2036580 or β-LGND. (b) TGFβ inhibits KLF15 mRNA and protein in cardiomyocytes, blocked by the p38 antagonist SB2036580 (0.1μM) (c) TAK1 activating phosphorylation is stimulated by AngII or TGFβ, inhibited by β-LGND. *p<0.05 vs. control, + p<0.05 for TGFβ or AngII vs same plus β-LGND, n=3 exps. (d) TAK1 siRNA diminishes TGFβ or AngII-stimulated p38α activity. The latter was seen as phosphorylation at tyrosine182. *p<0.05 vs control, +p<0.05 for TGFβ or AngII vs same + β-LGND, n=3 exps. TAK1 siRNA validation is also shown. (e) Flow cytometry analysis of β-LGND inhibition of phospho-kinases due to cAMP/PKA. *p<0.05 for control vs. AngII-stimulated phospho-TAK1, phospho-p38α, or KLF15 proteins. +p<0.05 for AngII vs AngII + β-LGND, ++p<0.05 for AngII + β-LGND vs same + either H-89 (PKA inhibitor) or RP-8-Br-cAMP (cAMP inhibitor), n=3 exps.

Article Snippet: Additional antibodies and phospho-specific antibodies used for immuno-blots were obtained from the followings: Cell Signaling Technology (Danvers, MA) TAK1 (D94D7) (#5206), Phospho-ATF-2 (Thr71) (#9221), Phospho-TAK1 (Thr187) (#4536); Santa Cruz, Biotechnology (Dallas, TX), KLF15 (A5) (SC-271675), GAPDH (0411) (sc-47724), MYH7 (A4.951) (sc-53090), Actin (2Q1055) (sc-58673), p38 Antibody (A-20) (sc-535), phospho-p38 (Thr 180/Tyr 182) (sc-17852-R); (Boster Biological Technology, Pleasanton, CA), ACTA2 (M01072–1).

Techniques: Activity Assay, Control, Phospho-proteomics, Biomarker Discovery, Flow Cytometry, Inhibition

Journal: Molecular and cellular endocrinology

Article Title: Estrogen receptor beta maintains expression of KLF15 to prevent cardiac myocyte hypertrophy in female rodents

doi: 10.1016/j.mce.2017.11.004

Figure Lengend Snippet: AngII acting through TGFβ stimulates a TAK1-p38α kinase axis that inhibits KLF15 expression and nuclear localization of the protein. This contributes to increased gene expression and cardiomyocyte hypertrophy. ERβ acting through protein kinase A opposes TAK1-p38α activation. This restores KLF15 abundance and nuclear localization, contributing in part to inhibition of AngII-induced gene expression and cardiomyocyte hypertrophy.

Article Snippet: Additional antibodies and phospho-specific antibodies used for immuno-blots were obtained from the followings: Cell Signaling Technology (Danvers, MA) TAK1 (D94D7) (#5206), Phospho-ATF-2 (Thr71) (#9221), Phospho-TAK1 (Thr187) (#4536); Santa Cruz, Biotechnology (Dallas, TX), KLF15 (A5) (SC-271675), GAPDH (0411) (sc-47724), MYH7 (A4.951) (sc-53090), Actin (2Q1055) (sc-58673), p38 Antibody (A-20) (sc-535), phospho-p38 (Thr 180/Tyr 182) (sc-17852-R); (Boster Biological Technology, Pleasanton, CA), ACTA2 (M01072–1).

Techniques: Expressing, Gene Expression, Activation Assay, Inhibition

Journal: Cancer discovery

Article Title: p38MAPK plays a crucial role in stromal mediated tumorigenesis

doi: 10.1158/2159-8290.CD-13-0743

Figure Lengend Snippet: (A) Western blot analysis demonstrating that SB203580 treatment inhibits p38MAPK activity. As expected, SB203580 treatment significantly impacts phosphorylation of p38MAPK’s direct downstream target, Hsp27. Total Hsp27 was used as loading control.

Article Snippet: The primary antibodies used were: polyclonal AUF1 (Millipore, Billerica, MA, catalog number 07260MI) at 1:3000, polyclonal p-p38 (PhosphoSolutions, Aurora, CO, catalog number p190-1802) at 1:1000, polyclonal p38 (Cell Signaling, Boston, MA, catalog number 9218) at 1:1000, monoclonal β-catenin (BD Biosciences, San Jose, CA, catalog number 610153) at 1:5000, and monoclonal α-tubulin (Abcam, Cambridge, MA, product number ab6160) at 1:1000.

Techniques: Western Blot, Activity Assay, Phospho-proteomics, Control

Journal: Cancer discovery

Article Title: p38MAPK plays a crucial role in stromal mediated tumorigenesis

doi: 10.1158/2159-8290.CD-13-0743

Figure Lengend Snippet: (A) RNA-seq analysis was performed on young fibroblasts, senescent fibroblasts, and senescent fibroblasts treated with SB203580. RNA-seq results were analyzed to determine the number of factors upregulated in response to senescence (SASP factors) and the number of p38MAPK-dependent factors. These results were also analyzed for overlap with the expression profiles of breast cancer (BC)-associated stroma.

Article Snippet: The primary antibodies used were: polyclonal AUF1 (Millipore, Billerica, MA, catalog number 07260MI) at 1:3000, polyclonal p-p38 (PhosphoSolutions, Aurora, CO, catalog number p190-1802) at 1:1000, polyclonal p38 (Cell Signaling, Boston, MA, catalog number 9218) at 1:1000, monoclonal β-catenin (BD Biosciences, San Jose, CA, catalog number 610153) at 1:5000, and monoclonal α-tubulin (Abcam, Cambridge, MA, product number ab6160) at 1:1000.

Techniques: RNA Sequencing, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Embryonic Hyperglycemia Delays the Development of Retinal Synapses in a Zebrafish Model

doi: 10.3390/ijms23179693

Figure Lengend Snippet: Synaptophysin accumulation in the OPL is reduced in 5 dpf hyperglycemic larvae. ( A ) Transverse retinal sections from 5 dpf control and hypoglycemic larvae were immunostained with fluorescently labeled antibodies specific for the synaptic vesicle marker synaptophysin. Maximal intensity projections are shown. Scale bar, 5 µm. Synaptophysin accumulation was reduced in hyperglycemic larvae relative to that in control larvae. ( B ) Quantitative analyses of IHC for synaptophysin accumulation in control and hyperglycemic larvae are indicated by box-and-whisker plots. Boxes indicate median value ranges, while whiskers indicate 5th–95th percentile values. Data represent the average value of the area of expression in 6–9 animals obtained from 3–5 independent experiments we sampled per condition, shown as individual data points. Scale bar, 5 µm. * p < 0.05; two-tailed t -test. Abbreviations used: dpf, days post-fertilization; SEM, standard error of the mean.

Article Snippet: Synaptophysin , Polyclonal anti-synaptophysin , Rabbit , 1:250 , Alomone lab (ANR-013) , Synaptic vesicles , [ ] .

Techniques: Labeling, Marker, Whisker Assay, Expressing, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Embryonic Hyperglycemia Delays the Development of Retinal Synapses in a Zebrafish Model

doi: 10.3390/ijms23179693

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: Synaptophysin , Polyclonal anti-synaptophysin , Rabbit , 1:250 , Alomone lab (ANR-013) , Synaptic vesicles , [ ] .

Techniques: Marker